Assembly of the Bacteriophage T4 Primosome

描述(由申请人提供)： 噬菌体T4复制系统是原核生物和真核生物DNA复制相关机制中研究最多的一种。这项提议的重点是DNA复制背景下的原始体的组装和功能。要完全理解这一点，就需要将原始染色体的各个蛋白质作为一个大型的酶机器来研究。原始体的组装过程将通过系综和单分子FRET平衡和预稳定状态的研究来进行。有关原始体的更多结构信息将通过电子显微镜获得。通过交联/足迹研究以及在活跃的转位和启动过程中使用FRET监测蛋白质-蛋白质的相互作用，将阐明原始体的功能。该项目的完成将导致对原始体的组装和功能的详细描述，类似于已经确定的聚合酶全酶。由于DNA复制过程对包括癌症在内的人类疾病有着深远的影响，了解DNA复制的保真度具有重大的人道主义和经济意义。

DESCRIPTION (provided by applicant): The bacteriophage T4 replication system is one of the best-studied of the related mechanisms of DNA replication employed by prokaryotes and eukaryotes. This proposal is focused on the assembly and functioning of the primosome in the context of DNA replication. A complete understanding will require studying the individual proteins of the primosome as a large enzyme machine. The assembly process of the primosome will be interrogated by ensemble and single-molecule FRET equilibrium and pre-steady state studies. Additional structural information on the primosome will be gained by electron microscopy. The functioning of the primosome will by elucidated by cross-linking/footprinting studies and by monitoring protein-protein interactions using FRET during active translocation and priming by the primosome. Completion of this project will result in a description of the assembly and functioning of the primosome in detail similar to that already determined for the polymerase holoenzyme. Since the process of DNA replication has profound implications for human disease, including cancer, understanding the fidelity of DNA replication is of significant humanitarian and economic interest.